The field of the invention is immunophilins.
FK506, rapamycin, and cyclosporine A (CsA) are immunosuppressive agents recently shown to inhibit signal transduction pathways following binding to cytoplasmic receptors termed immunophilins (Schreiber, Science 251:283-287, 1991). When complexed with an immunophilin, each of FK506 and CsA apparently interferes with cytoplasmic signaling pathways leading to transcription in the T cell (Dumont et al., J. Immunol 144:251-258, 1990; Bierer et al., Proc. Natl. Acad. Sci. U.S.A. 87:9231-9235, 1991). In contrast, rapamycin bound to an immunophilin inhibits a lymphokine-dependent signaling pathway in the T cell that, if not so inhibited, leads to proliferation of the cell.
The predominant FK506- and rapamycin-binding immunophilin in human T cells is the 12 kDa cytosolic receptor variously termed "FKBP" or "HCB" (and hereinafter referred to as FKBP-12) (Harding et al., Nature 341:758-760, 1989; Siekierka et al., Nature 341:755-757, 1989; European patent application publication number 0379342: each of which publications is herein incorporated by reference). Each of the latter two references sets forth the nucleotide sequence of the cDNA encoding FKBP-12, as well as the deduced amino acid sequence. Although FKBP-12 catalyzes the interconversion of cis and trans rotamers of peptidyl-prolyl amide bonds of peptides, and binding to FK506 or rapamycin inhibits this activity of FKBP-12, inhibition of this rotamase activity was shown to be insufficient for mediating the actions of FK506 and rapamycin in T cells (Bierer et al., Proc. Natl. Acad. Sci. U.S.A. 87:9231-9235, 1990; Bierer et al., Science 226:556-559, 1990). Recent studies show that the same is true of the rotamase activity of human cyclophilin (Sigal et al., J. Exp. Med. 173:619-628, 1991), which is the predominant CsA-binding protein in human T cells (Handschumacher et al., Science 226:544-546, 1984).
Van Duyne et al. (Science 252:836, 1991) have published an x-ray crystallographic study of FKBP-12 complexed with FK506. The following passages and the enclosed FIGS. 7, 8A and 8B are taken from pages 840-841 of this article (citations inserted):
The protein component . . . is a five-stranded antiparallel .beta. sheet wrapping with a right-handed twist around a short .alpha. helix. The five-stranded antiparallel .beta.-sheet framework includes residues 2 to 8, 21 to 30, 35 to 38 with 46 to 49, 71 to 76, and 97 to 106 (Surface areas and secondary structure were calculated with the program DSSPNOV (Kabecha and Sander, Biopolymers 22:2577, 1983) with topology +3, +1, -3, +1 (Richardson, Nature 268:495, 1977). The .alpha. helix is formed by residues 57 to 63. Thus, the fold of the protein is identical to that independently seen in the solution structure of uncomplexed FKBP (Michnick et al., Science 252:836, 1991). PA1 FK506 binds in a shallow cavity between the .alpha. helix and the .beta. sheet, with roughly 430 .ANG..sup.2 (50%) of the ligand surface being buried at the protein-ligand interface and the remainder, encompassing the region around the allyl and the cyclohexyl groups, being exposed to solvent. Loops composed of residues 39 to 46, 50 to 56, and 82 to 95 flank the binding pocket, which is lined with conserved, aromatic residues. The side chains of Tyr.sup.26, Phe.sup.46, Phe.sup.99, and Val.sup.55 -Ile.sup.56 make up the sides of the pocket, while the indole of Trp.sup.59, in the .alpha. helix, is at the end of the pocket and serves as platform for the pipecolinyl ring, the most deeply buried part of FK506 . . . Both the location and the orientation of the pipecolinyl ring are consistent with NOEs observed between FK506 and Trp.sup.59, Phe.sup.46, and Tyr.sup.26 (Wandless et al., J. Am. Chem. Soc. 113:2339, 1991). As might be expected, there are no water molecules in the hydrophobic binding pocket. PA1 There are five hydrogen bonds between FKBP and FK506: Ile.sup.56 -NH to C-1 lactone carbonyl, Glu.sup.54 -CO to C-24 hydroxyl, Gln.sup.53 -CO to C-24 hydroxyl (through a water molecule), Asp.sup.37 -CO.sub.2.sup.- to C-10 hemiketal hydroxyl, and Tyr.sup.82 -OH to C-8 amide oxygen. The first three, involving residues near the NH.sub.2 -terminus of the helix, form an array reminiscent of the antiparallel sheet interactions in many peptide-protein (especially protease) complexes, suggesting that the region of FK506 spanning C-24 to C-1 through the lactone linkage may mimic a dipeptide. As has been noted (Albers et al., J. Org. Chem. 55:4984, 1990), the adjacent pyranose-pipecolinyl region also resembles a dipeptide, and thus FK506 may prove to be an illustrative example of extended peptidomimicry. The fifth hydrogen bond, involving the C-8 amide, is the most conspicuous because it is nearly orthogonal to the carbonyl plane and thus may be relevant to the mechanism of rotamase activity. PA1 Protein-protein hydrogen bonds help maintain the organization of the binding pocket, particularly in restraining the flexible loops, which assume well-defined conformations in the complex. For instance, Asp.sup.37 -CO.sub.2.sup.- forms hydrogen bonds not only with the C-10 hemiketal hydroxyl of FK506, but also with the Arg.sup.42 and Tyr.sup.26 side chains (Horovitz et al., J. Mol. Biol. 216:1031, 1990). Two well-ordered water molecules bridge the loop from Tyr.sup.82 to Ala.sup.95 by mediating hydrogen bonds from Tyr.sup.82 -NH to Ala.sup.95 -CO and from Gly.sup.83 -CO to Pro.sup.92 -CO. Three residues within this loop, Tyr.sup.82, His.sup.87, and Ile.sup.91, contact FK506, with the side chains of the latter two forming a surface complementary to the pyranose methyl group region. The observed loop geometry thus plays a major role in ligand binding, but it also forces Ala.sup.81 to adopt unfavorable .phi., .PSI. values of -141.degree. and -120.degree. respectively.
A number of cyclophilins associated with subcellular organelles have been reported. These include the cyclophilin-like protein encoded by the gene nina A of Drosophila melanogaster (Shieh et al., Nature 338:67-70, 1989; Schreuwly et al., Proc. Natl. Acad. Sci. U.S.A. 86:5390-5394, 1989). The Nina A gene product has an N-terminal signal sequence and a C-terminal hydrophobic domain that may serve as a membrane anchor. Mutations in this gene result in photoreceptor cell dysfunction that may be due to improper trafficking of rhodopsin molecules. A yeast cyclophilin with a signal sequence has also been described (Koser et al., Nucleic Acids Res. 18:1643, 1990), and recently a human cyclophilin with a signal sequence and with sequence similarity to this yeast cyclophilin has been cloned (Price et al., Proc. Natl. Acad. Sci. U.S.A. 88:1903-1907, 1991).
FKBP-12 is the protein responsible for mediating the potent anti-proliferative actions of rapamycin in yeast. Deletion of the FKBP-12 gene in Saccharomyces cerevisiae results in rapamycin-resistant strains of yeast; rapamycin sensitivity is returned following transfection of either yeast or human FKBP-12 into the rapamycin-resistant mutant cells (Koltin et al., Mol. Cell. Biol. 11:1718-1723, 1991). Similar findings can be inferred from the studies of cyclophilin in two lower eukaryotes (Tropschug et al., Nature 342:953-955, 1989).